After erythrocytes, platelets are the most prevalent blood

Transcription

1 Review Circulating Platelets as Mediators of Immunity, Inflammation, and Thrombosis Milka Koupenova, Lauren Clancy, Heather A. Corkrey, Jane E. Freedman Abstract: Platelets, non-nucleated blood components first described over 130 years ago, are recognized as the primary cell regulating hemostasis and thrombosis. The vascular importance of platelets has been attributed to their essential role in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism. Increasing knowledge on the platelets role in the vasculature has led to many advances in understanding not only how platelets interact with the vessel wall but also how they convey changes in the environment to other circulating cells. In addition to their welldescribed hemostatic function, platelets are active participants in the immune response to microbial organisms and foreign substances. Although incompletely understood, the immune role of platelets is a delicate balance between its pathogenic response and its regulation of thrombotic and hemostatic functions. Platelets mediate complex vascular homeostasis via specific receptors and granule release, RNA transfer, and mitochondrial secretion that subsequently regulates hemostasis and thrombosis, infection, and innate and adaptive immunity. (Circ Res. 2018;122: DOI: /CIRCRESAHA ) Key Words: blood platelets communication hemostasis immunity infection thrombosis After erythrocytes, platelets are the most prevalent blood component, and they vary in size from 2 to 5 μm. 1 Once released by their megakaryocytic precursor, platelets enter the bloodstream and circulate for 7 to 10 days. 2,3 Platelets are complex cells that contain 3 distinct types of granules: α-granules, dense or δ-granules, and lysosomes. 4,5 Platelet α-granules contain various proteins, chemokines, cytokines, and growth factors that are assembled in platelets by megakaryocytes and are necessary for normal platelet functionality. Platelet δ-granules contain small molecules such as ADP, serotonin, polyphosphates, glutamate, histamine, and calcium that are necessary for hemostasis. 4,6 Platelet lysosomes contain enzymes such as glycohydrolases and enzymes that degrade GPs (glycoproteins), glycolipids, and glycosaminoglycans. 4,7 Platelets do not have a nucleus but contain prepackaged proteins and various forms of RNA from their precursor cells. Found throughout the vasculature, platelets respond to signals from the endothelium, circulating cells, or other blood components. In this review, we will focus on the complex roles of platelets in thrombosis and immunity facilitated by their various interactions with the endothelium and circulating immune cells. Established Mechanisms for Hemostasis and Thrombosis It is well established that in primary hemostasis, platelets adhere to the damaged vessel wall at the site of injury. 2,3,8 This process occurs through multiple signaling cascades and is highly dependent on GPs expressed on the platelet surface. 2,3,8 The inhibitory function of the intact endothelium (specifically, the production of prostacyclin and nitric oxide as well as expression of CD39) is decreased on vessel damage, and extracellular matrix proteins such as collagen are exposed to the circulation. 2,3 Platelet adhesion, or initial binding of platelets to the damaged site, differs depending on the level of shear stress in the circulation. At high shear stress, circulating plasma-derived vwf (von Willebrand factor) rapidly unfolds and deposits on the exposed subendothelial collagen at the injury site. The unfolding of vwf exposes multiple binding sites for GP1b (CD42), a member of the GP1b-V-IX receptor complex. 2,3,8 This process decreases platelet velocity allowing for low shear stress and GPVI collagen interactions. 2,3,8 Under low shear stress, platelets adhere predominantly to collagen through their GP1a/2a integrins. This phase of adhesion leads to platelet activation and many distinct platelet physiological and cytoskeletal changes, including a shift from a resting discoid shape to an activated state with numerous pseudopodia (Figure 1). 2,3,8 The initial adhesion and activation of platelets is followed by recruitment of additional platelets from the circulation and formation of 3-dimensional aggregates through several molecular interactions. The recruitment of other platelets, their activation, and platelet aggregation is mediated by platelet generation of thromboxane A2 and release of ADP from their δ-granules. Platelet platelet interactions during thrombi formation are further mediated and stabilized by the platelet fibrinogen receptor GP2b/3a. 2,3,8 Depending on the level of the injury, blood can leak into the tissue and expose platelets to tissue factor expressed by cells that comprise the vessel. The presence of tissue factor leads to thrombin generation which mediates firm platelet aggregation. From the Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester. Correspondence to Milka Koupenova, PhD, University of Massachusetts Medical School, Albert Sherman Center, 368 Plantation St, AS7-1014, Worcester, MA milka.koupenova@umassmed.edu 2018 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 338 Circulation Research January 19, 2018 Nonstandard Abbreviations and Acronyms ApoE apolipoprotein E COX cyclooxygenase CTAP-3 connective tissue activating peptide 3 CX3CR1 C-X3-C motif chemokine receptor 1 CXCL7 chemokine (C-X-C motif) ligand 7 CXCR1/2 C-X-C chemokine receptor type 1 or 2 DCs dendritic cells E-selectin endothelial selectin FcγRIIa Fc fragment of IgG receptor IIa GM-CSF granulocyte macrophage colony-stimulating factor GP glycoprotein HNP1 human neutrophil peptide 1 HUVEC human umbilical vein endothelial cells ICAM-1 intercellular adhesion molecule 1 IFN-γ interferon-γ IL interleukin JAM-C junctional adhesion molecule C MAC-1 macrophage-1 antigen (also integrin αmβ2 or CD11b/ CD18) MCP-1 monocyte chemotactic protein 1 (also CCL2) NET neutrophil extracellular trap NK natural killer P-selectin platelet selectin P2Y12 purinergic receptor 2Y12 PAR4 protease-activated receptor-4 PECAM platelet endothelial cell adhesion molecule (also CD31) PF4 platelet factor 4 (also CXCL4) PSGL1 P-selectin glycoprotein ligand 1 RANTES regulated on activation, normal T cell expressed and secreted (also CCL5) spla2-iia serum secretory phospholipase A2-IIa TGF-β transforming growth factor-β TLR Toll-like receptor TNFRII tumor necrosis factor receptor 2 Tβ-4 thymosin β-4 VCAM-1 vascular cell adhesion molecule 1 VE-cadherin vascular endothelial cadherin VEGF vascular endothelial growth factor vwf von Willebrand factor Once formed, the primary hemostatic plug retracts and becomes firmly anchored at the site of injury, preventing rebleeding. Clot stabilization is described as secondary hemostasis and is characterized by consolidation of the platelet mass through actin/myosin-mediated platelet retraction. At this stage, thrombin, in addition to activating platelets, converts fibrinogen to fibrin leading to the formation of a network of fibrin fibers. In certain settings, normal hemostasis is overwhelmed by pathological factors leading to uncontrolled clot formation and potential vessel occlusion. As previously discussed, uncontrolled clot formation is defined as thrombosis and, in the artery, is primarily mediated by platelets. Arterial thrombosis is a central cause of myocardial infarction and stroke (embolic or in situ), whereas venous clot formation may lead to deep vein thrombosis and/or venous thromboembolism. In response to blood-borne pathogens and consequent tissue damage, platelets are also involved in a coordinated intravascular coagulation response recently termed immunothrombosis. During this process, platelets and immune cells form a physical barrier of confinement preventing dissemination of pathogens and potentially leading to the activation of the innate and adaptive branches of the immune system. 6,9 Interestingly, platelets mediate the crosstalk between the hemostatic and the immune system using similar pathways (Table 1). Consistently, various viral or bacterial infections have been found to contribute to the risk of thrombosis that manifests as arterial thrombosis or venous thromboembolism and may contribute to atherosclerosis Thus, although immunothrombosis may be an efficient way of assisting the immune system, it may significantly contribute to the overall risk of cardiovascular disease. Contemporary Mechanisms for Hemostasis and Thrombosis Recent in vivo studies have provided a more complex view of hemostasis and thrombosis. This new model suggests that the hemostatic plug is characterized by a stringent architecture, with a distinct core and outer shell. 40,41 Fibrin deposition is distinctly localized at the base of the core in the extravascular space before complete hemostasis is achieved. A platelet activation gradient is also established, with the inner core of the plug composed of tightly packed, activated platelets which are degranulated and P-selectin (platelet selectin) positive, whereas the outer shell is composed of less activated loosely packed platelets that do not express P-selectin. 40,41 The outer shell, however, is stable and permeable to plasma solutes. Consistent with the activation gradient of platelet distribution, there is a distinct distribution of platelet agonists throughout the thrombus. The core of the plug contains a high concentration of thrombin and, as the plug becomes more porous, a gradient of ADP and thromboxane A2 develops. 40,41 The porous outer shell of the thrombus may allow for recruitment of leukocytes necessary for injury repair or pathogen elimination. At the site of injury in veins (low shear stress), in addition to platelet activation and thrombus formation, thrombin plays a central role in leukocyte recruitment to the hemostatic plug. This process is mediated predominantly by activated platelets and not endothelial cells. 10 Activation/cleavage of the platelet thrombin receptor PAR4 (protease-activated receptor-4) promotes leukocyte recruitment and migration, 10 and platelet P-selectin enables leukocyte interaction with the thrombus. 10,42 Thrombin also mediates fibrin generation which limits leukocyte migration in the clot by forming a physical barrier. 10 Finally, platelet GP1b can bind thrombin depleting its effect and limiting leukocyte trafficking into the thrombus. 10 Under lower shear stress conditions either in veins or in arteries (ischemia reperfusion), platelet adhesive interactions can also lead to formation of a CXCL7 (chemokine (C-X-C motif) ligand 7) chemotactic gradient within the thrombus body. This gradient guides intravascular migration of leukocytes through the thrombi to the sites of injury via their CXCR1/2 (C-X-C chemokine receptor type 1 or 2) receptors. 42 This suggests that platelets, by mediating hemostasis, can also facilitate leukocyte recruitment to the clot enabling close contact of the immune system with potential pathogens

3 Koupenova et al Platelets and Vascular Communication 339 Figure 1. Differences in platelet thrombotic vs immune activation visualized by scanning electron microscopy. Isolated (A) human platelets or (B) human platelets mixed with leukocytes were treated with immune or thrombotic agonists. A, B, Varying levels of activation when platelets are activated via immune vs thrombotic pathways. Bar represents 4 μm. EMCV indicates encephalomyocarditis virus; Loxo, loxoribine; and TLR, Toll-like receptor. Reprinted from Koupenova et al 34 with permission. Copyright 2014, the American Society of Hematology. that may have penetrated the skin barrier. Further studies are necessary to elucidate how platelet distribution changes in vivo in pathogen-associated venous thrombi. Platelets and Endothelial Cells The interaction between platelets and endothelial cells has been previously and extensively reviewed. 11,43 Platelet rolling over intact endothelium is observed predominantly in veins and increases with endothelial activation in response to inflammatory stimulus or infection. Under low shear stress in veins, platelet endothelial interactions are mediated by platelet GP1bα and endothelial vwf. 11 Under higher shear stress in small venules, endothelial P-selectin and platelet PSGL1 (P-selectin glycoprotein ligand 1) or GP1bα mediate platelet rolling. 11 In the absence of additional signals, platelets disengage and return to the circulation. When the endothelium is under stress, firm adhesion of platelets will occur through endothelial ICAM-1 (intercellular adhesion molecule 1; or through endothelial αvβ3) and platelet GP2b/3a in a fibrinogen-dependent manner. Endothelial stress is manifested during inflammation or infection by an increase in endothelial surface expression of P-selectin, E-selectin (endothelial selectin), VCAM (vascular cell adhesion molecule), and ICAM Endothelial cells also regulate platelet activation and thrombus size by secreting thromboregulators such as nitric oxide (NO) and prostacyclins and removing ADP/ATP through CD39 ectonucleotidase. NO can also be secreted by platelets and can inhibit additional platelet recruitment, reduce P-selectin expression, and promote disaggregation. 45 In addition to integrin/selectin surface expression, platelets can also affect the endothelium by increasing their surface expression of CD154 (CD40L) or by secreting cytokines such as IL (interleukin)-1β. Seconds after thrombin stimulation of human platelets in vitro, CD154 surface expression occurs. Platelet CD154, in turn, can lead to expression of endothelial E-selectin, VCAM-1, and ICAM-1, as well as secretion of MCP-1 (monocyte chemotactic protein 1) and IL-8 from endothelial cells. 35 In addition to surface protein expression, platelets can synthesize proteins from stored RNA templates. 46,47 One of these proteins, the cytokine IL-1β, is synthesized in response to inflammatory stimuli 46 (Figure 2). Released IL-1β can increase endothelial permeability, as well as recruitment and attachment of leukocytes to the endothelium. 48,49 This leads to platelet alteration of the inflammatory cell transmigration to damaged or infected tissue. Interestingly, IL-1β increases the binding potential of platelets to collagen and fibrinogen and, in the presence of collagen, promotes aggregation. 50 Receptors in Platelet Immune Cell Interactions To understand the role of platelets in thrombosis in diverse clinical settings, their role in immunity should be addressed. Under certain infectious conditions or inflammatory stimuli, platelets directly interact with circulating leukocytes by changing surface expression of P-selectin or CD40. 5 Platelet P-selectin and CD40 are recognized by leukocyte PSGL1 and CD154, respectively, leading to the formation of platelet leukocyte aggregates. Platelets form these aggregates predominantly with circulating monocytes or neutrophils, thereby contributing to the innate immune response. 5 Platelets also contribute to adaptive immunity by interacting with and activating dendritic cells (DCs) through the CD40 CD154 axis. Platelet-mediated activation of DCs causes the DCs to present antigen to T cells. 5 In addition, platelet activation can also lead to the release of δ-granule content and secretion of molecules such as serotonin and RANTES (regulated on activation, normal T cell expressed and secreted; CCL5) that are also known to mediate T-cell activation and differentiation. 23,29,30 Notably, platelets contain the mrna transcripts for all TLR1 to TLR10 (Toll-like receptors). 4,51,52 TLRs are pathogen-associated molecular pattern recognition receptors that are key regulators in the initiation of the innate immune response to foreign organisms. Platelets express functional TLR4, TLR2, and TLR9, 59,60 and these TLRs can initiate thrombotic responses of varying intensities (Table 2). Platelet TLR4 and platelet TLR2 can also mediate interactions with neutrophils. 54,57 In addition, platelets express functional TLR7 34 and TLR3 62,63 ; however, these TLRs do not induce prothrombotic responses (aggregation mediated by thrombin 34 ), but instead alter platelet leukocyte interactions. Stimulation of TLR7 leads to the platelet surface expression of P-selectin and CD154, suggesting select α-granule release. 34 Stimulation of TLR3 does not lead to P-selectin or CD154 surface expression but, at high agonist concentration (possibly equivalent to late stages of infection), potentiates arachidonic acid mediated, ADPmediated, or collagen-mediated aggregation. 62,63 Interestingly,

4 340 Circulation Research January 19, 2018 Table 1. Platelet-Derived Mediators Linking Thrombosis, Infection, and Immunity Category of Platelet Mediator Integrins α-granule proteins δ-granule molecules Surface protein expression Synthesized/secreted Platelet Receptor/; Protein/Molecule GP1b (Cd42) GP1a/2a GP2b/3a (CD41) PF4 (CXCL4) RANTES (CCL5) Vascular or Circulating Cell Interaction Setting Functions Ref. vwf; endothelial cells; leukocytes; bacteria Subendothelial collagen Other platelets through Fgn; T cells; bacteria T cells; monocytes; RBCs; bacteria immunity; atherogenesis High shear stress/ infection Unfolded vwf deposited on collagen; endothelial P-selectin; binds thrombin limiting leukocyte recruitment; binds complement C3 on bacteria to enhance adaptive immunity Low shear stress Collagen 8 Hemostasis; infection; immunity Infection; atherosclerosis infection; chronic inflammation TGF-β Tumor cells (TGF-βR) Metastasis β-defensin Serotonin (5HT) Neutrophils Endothelial cells; T cells Staphylococcus aureus α-toxin Hemostasis/ thrombosis; adaptive immunity ADP Platelet P2Y12; P2Y1 Hemostasis P-selectin Leukocyte PSGL1 (neutrophils, monocytes, DC); endothelial PSGL1; metastatic cells PSGL1 Infection; other platelets PSGL1 Endothelial P-selectin High shear stress CD40 Leukocyte CD154 Inflammation/ infection/immunity CD154 Endothelial CD40 Inflammation/infection IL-1β TxA2 Endothelial IL-1R associated with αvβ3 in the presence of Fgn Thromboxane A2 Receptor Infection; inflammation Hemostasis Platelet aggregation; endothelial ICAM-1 or αvβ3 leading to firm adhesion; aggregation with activated T-cytolytic and T-helper cells; aggregation around bacteria; increase after H1N1 infection Limit Th17 expansion and differentiation; monocyte recruitment (heterodimer with RANTES); inhibits TGF-β signaling; binds Gram-negative bacteria increasing opsonization; kill plasmodium in RBCs T-cell activation and differentiation; monocyte/ macrophage adhesion and recruitment; Reduce NK antitumor activity; contribute to induction of invasive epithelial mesenchymal transition to metastasis , ,23, Netosis 28 Constricts injured blood vessels; enhances platelet aggregation to minimize blood loss; T-cell activation and differentiation; endothelial cell proliferation Platelet recruitment, activation, and aggregation during clot formation; exposure of P-selectin (P2Y12, P2Y1) and PS and thrombin generation (P2Y12) Platelet neutrophil and platelet monocyte HAGs; interactions of leukocytes with the thrombi; platelet DC interactions; increase as a result of TLR7 stimulation; metastatic PSGL1 adhesion Platelet endothelial interactions for thrombus formation in small venules Surface expression as a result of TLR7 platelet neutrophil tethering to the endothelium; platelet DC leading to T-cell antigen presentation Increase in endothelial expression of E-selectin, VCAM-1, and ICAM-1, as well as secretion of MCP-1 and IL-8 29, ,6,10,32, ,34 Increases endothelial permeability by secreting NO 36 Platelet recruitment, activation, and aggregation during clot formation CXCL indicates chemokine (C-X-C motif) ligand; DC, dendritic cells; Fgn, fibrinogen; GP, glycoprotein; HAG, heterotypic aggregates; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MCP, monocyte chemotactic protein; NK, natural killer cell; NO, nitric oxide; P-selectin, platelet selectin; P2Y12, purinergic receptor 2Y12; PF, platelet factor; PS, phosphatidylserine; PSGL1, P-selectin glycoprotein ligand 1; RANTES, regulated on activation, normal T cell expressed and secreted; RBC-red blood cell; TGF-β, transforming growth factor-β; Th17, T helper 17 cells; TLR, Toll-like receptor; TxA2, thromboxane A2; VCAM-1, vascular cell adhesion molecule 1; and vwf, von Willebrand factor. 35 5

5 Koupenova et al Platelets and Vascular Communication 341 Figure 2. Platelet-mediated interactions with vascular or circulating cells. Platelets interact with endothelial and immune cells in the circulation, orchestrating a response to microbes, inflammatory stimuli, and vessel damage. Through their TLRs (Toll-like receptors; or inflammatory signals), platelets can change their surface expression and release their granule content, thereby engaging different immune cells. Platelets form heterotypic aggregates (HAGs) and initiate innate immune responses in the presence of TLR agonists and viruses such as encephalomyocarditis virus (EMCV), coxsackievirus B (CVB), dengue, flu, HIV. Platelets can interact with dendritic cells (DC) through their P-selectin (platelet selectin), activate them to become antigen (Ag) presenting through their CD154. By releasing α- or δ-granule content which leads to IgG (IgG1, IgG2, IgG3) production and control of T-cell function, platelets engage the adaptive immune response. Similarly, platelets are able to activate the endothelium, make it more permeable, and mediate leukocyte trafficking to the inflamed endothelium. Proteins in bold represent changes of expression on the platelet surface. Continuous lines represent direct binding; dotted lines represent interaction through secretion. 5HT indicates serotonin; CMV, cytomegalovirus; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; PF4, platelet factor 4; PSGL1, P-selectin glycoprotein ligand 1; RANTES, regulated on activation, normal T cell expressed and secreted; TGF-β; transforming growth factor-β; and VCAM-1, vascular cell adhesion molecule 1. analysis of platelets from 1864 people shows that women have significantly higher expression of all TLRs when compared with men. 51 TLR expression in platelets from women associated with an increase of plasma P-selectin, whereas, in men, TLR expression associated with an increase in IL-6, TNFRII (tumor necrosis factor receptor 2), and soluble ICAM Importantly, platelet immune activation differs from hemostatic, thrombin-mediated platelet responses. Scanning electron micrographs of human platelets alone and in the presence of leukocytes illustrate a distinct morphological shape change dependent on their function (Figure 1). 34 Platelet hemostatic function demonstrated by stimulation with thrombin causes complete disappearance of the platelet s discoid shape and conversion into long intertwined spaghetti-like pseudopodia (Figure 1). Platelet immune function demonstrated by stimulation with TLR7 agonist or TLR7-activating virus leads to smaller platelet groups in which single platelets can be identified with fewer pseudopodia connecting to other platelets or to leukocytes (Figure 1). This suggests that platelets undergo distinct forms of activation depending on the stimulatory signal and their functional role. Different levels of activation seem necessary for the proper formation of either the hemostatic plug or recruitment of leukocytes (see Contemporary Mechanisms for Hemostasis and Thrombosis). Platelets and Neutrophils The platelet interaction with neutrophils is central to initiating the immune response (Figure 2). In humans, neutrophils are the most abundant blood leukocyte and, together with monocytes, are the major initiators of the innate immune response. Platelets form heterotypic aggregates with neutrophils as a function of TLR7, TLR2, and TLR4 activation in the circulation. 34,54,57 Platelet neutrophil interactions are mediated by the platelet P-selectin/neutrophil PSGL1 axis. Neutrophils can also phagocytose activated platelets in vivo with internalization mediated by phosphatidylserine platelet surface expression. 65 Interestingly, as a result of TLR7 stimulation, platelet components (as measured by CD41) are internalized by neutrophils. 34 It is currently unclear whether platelet microparticles 66 or the entire platelet is internalized as a function of TLR7 stimulation. 34

6 342 Circulation Research January 19, 2018 Table 2. Platelet TLRs and Their Contribution to Thrombosis and Immunity TLRs TLR2 TLR4 TLR3* TLR7 TLR9 Agonist PAM 3 CSK 4 ; oxpc CD36 LPS pic; pau Loxoribine; R837; R848 CpG; Carboxyalkylpyrrole protein adducts (CAPA) Interaction With Cells Result Thrombosis Neutrophils Neutrophils, monocytes Neutrophils Platelet neutrophil HAGs through P-selectin; platelet TLR2 contributes to netosis; activation of GP2b/3a; hyperreactivity and the prothrombotic state in the setting of hyperlipidemia Platelets reduce the time of netosis in vivo; platelets aggregate in TLR4/MyD88- and cgmp/pkg-dependent pathway Activation of NF-κB, PI3K-Akt, and ERK1/2 pathways in platelets; at high agonist concentrations may potentiate platelet aggregation; liver netosis (pic) Modest increase of P-selectin and CD40 through AKT and p38-mapk; no effect on platelet aggregation; liver netosis (R838) Platelet clumping in presence of collagen (CpG); platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway (CAPA) Platelet TLR Contribution to Netosis Organ Netosis Ref. + +? 56,57, ,54,136? + 62,63? + 34,64?? 59,60 *TLR3 may potentiate platelet aggregation at very high concentrations (50 μg/μl); Mitochondrial DNA can mediate netosis through TLR9. AKT indicates protein kinase B; CpG, 5 -cytosine-phosphate-guanine-3 ; ERK1/2, extracellular signal-regulated kinase 1 and 2; HAG, heterotypic aggregates; LPS, lipopolysaccharides; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; P-selectin, platelet selectin; pau, polyadenylic polyuridylic acid; pic, polyinosinic-polycytidylic acid; pic-poly(i:c); pau, poly(a:u); and TLR, Toll-like receptor. +indicates mediates;, does not mediate; and?, not known (up to date). Platelet neutrophil interactions are also important for neutrophil extracellular trap (NET) formation, a process termed netosis whereby pathogens are captured and neutralized Staphylococcus aureus α-toxin, for instance, mediates secretion of β-defensin 1 from platelets which can lead to NET formation. 28 In the setting of lipopolysaccharide activation of TLR4, platelets contribute to netosis by reducing the time required for NET formation 54 (Table 2). Platelet activation of TLR2 by PAM 3 CSK 4 also contributes to netosis (Table 2). Although various TLRs are implicated in organ netosis, the role of platelet TLRs, with the exception of TLR4, has not been elucidated (Table 2). Platelets and Monocytes Platelets can interact with monocytes as a consequence of inflammatory stimuli or infection (Figure 2). Platelet monocyte aggregates may be a more sensitive method for assessing platelet activation when compared with P-selectin expression. 70 Circulating platelet monocyte aggregates are elevated in patients with acute myocardial infarction, 71,72 and, in older patients, platelet monocyte aggregates are associated with vascular thromboembolism. 73 Murine studies have shed light on the complex interaction between platelets and monocytes. During chronic inflammation, simultaneous activation of platelets and neutrophils leads to secretion of RANTES and HNP1 (human neutrophil peptide 1), respectively. 24 These proteins form heterodimers responsible for monocyte/macrophage adhesion and recruitment in a model of myocardial infarction. 24 Thrombin-activated human platelets also express the chemokine receptor CX3CR1 (C-X3-C motif chemokine receptor 1) leading to the formation of platelet monocyte complexes through the CX3CL1 ligand on monocytes. 74 Platelets and Eosinophils Platelets are described to affect eosinophil function as well. Eosinophils are granulocytes that compose 2% of all leukocytes, and it has been proposed that they may contribute to the innate immune response to helminth parasite (worms) infection. Pathogenically, eosinophil function has been attributed to the profound damage they cause in an allergic asthma. Direct interaction between platelets and eosinophils in the circulation during helminth infection in vivo has not been described. However, coincubation experiments of platelets and eosinophils (for 48 hours) show that platelets prolong eosinophil life by secreting GM-CSF (granulocyte macrophage colony-stimulating factor). 75 This suggests that platelets may enable eosinophils in fighting helminth infection. However, by delaying eosinophil apoptosis platelets may also contribute to asthma-mediated tissue damage. A murine model of allergic inflammation has suggested that platelets through their P-selectin recruit eosinophils to the lungs 76 and consequently may propagate tissue damage. Allergen-sensitized mice had an increase of platelet eosinophil aggregates in the circulation and in the lung tissue, and aggregate interactions were mediated through platelet P-selectin and eosinophil PSGL1. 76 Platelets, B Cells, and T Cells When interacting with lymphocytes, platelets are more selective and bind preferentially to larger, activated cells. 13 In the presence of lipopolysaccharide or ADP, aggregation between platelets and B cells is almost unaffected. However, when platelets are coincubated with B cells for 3 days in vitro, B cells increase their production of IgG1, IgG2, and IgG3, suggesting that platelet content can contribute to B-cell function and alter adaptive immunity. 77

7 Koupenova et al Platelets and Vascular Communication 343 T-cell activation more robustly increases platelet aggregation via both T-cytolytic and T-helper cells 13 (Figure 2). This process is mediated by platelet GP2b/3a, CD154, and lymphocyte CD11b. 13 CD154 is expressed under various stimulants (such as TLR7 activation 34 ) and is capable of enhancing T-cell immunity to viral challenge. Evidence suggests that, by interacting with lymphocytes, platelets may facilitate the recruitment of these cells to an injured vessel at a site of inflammation or infection, 78 and this is a central step in T-cell trafficking. Activated platelets may mediate T-cell functions further by releasing many factors, such as PF4 (platelet factor 4; CXCL4) RANTES or serotonin. 16,29,30 The release of these factors impacts T-cell function in various ways. PF4, for instance, is necessary for the limitation of Th17 (T helper 17 cells) expansion and differentiation. 17 Serotonin, the largest peripheral circulation reservoir of which is stored in platelet δ- granules, can push naive T cells to activate and proliferate. 29,30 Platelets and DCs As previously mentioned, platelets interact with and influence another important cell in adaptive immunity, the DC. Platelets, through the CD40 CD154 axis, can induce DCs to present antigen to T cells. 5 In addition, platelet DC interactions can be mediated by the P-selectin/PSGL1 axis, followed by firm adhesion through platelet JAM-C (junctional adhesion molecule C) with MAC-1 (macrophage-1 antigen) on DCs 32 (Figure 2). Interestingly, this recruitment and activation of DCs is followed by phagocytosis of platelets by DCs. 32 The mechanism by which DCs change after platelet phagocytosis is still largely unknown. It is possible also that serotonin originating from phagocytosed platelets may be used by DCs and released as needed for T-cell proliferation and differentiation. Platelets and Natural Killer Cells The interaction between platelets and natural killer (NK) cells is the least understood interaction within the circulation (Figure 2). In murine studies, tumors coated with platelets become impermeable to NK-cell destruction 79 with fibrinogen involvement. 80 It has been proposed that circulating metastatic cells attract platelets and influence them to release their granule content. As a result, human NK cells lose their cytotoxicity and their ability to produce IFN-γ (interferon-γ), possibly through the downregulation of NK G2D ligand mediated by platelet TGF-β (transforming growth factor-β) release. 25 However, the baseline communication between platelets and NK cells remains unknown as does the effect on this interaction during infection or autoimmune disease. Platelet Leukocyte Interactions in Atherogenesis The role of platelet leukocyte interactions during inflammation has been determined to be contributory in murine models of atherosclerosis. It is now established that activated platelets can increase endothelial atherogenic potential and lead to elevated platelet leukocyte aggregate formations in the circulation. Platelets may further contribute to atherogenesis through the delivery of proinflammatory factors to leukocytes and endothelial cells. 81 The platelet-derived factors implicated in this setting are surface P-selectin and the platelet-derived chemokines RANTES and PF4. 81 PF4/RANTES heterodimer disruption reduces atherosclerosis in ApoE (apolipoprotein E)- deficient mice by attenuating monocyte recruitment. 16 Murine models lacking P-selectin show that this adhesion receptor is a key mediator of leukocyte recruitment into lesions and promotes their advancement in ApoE-deficient mice. 82 In this case, both platelet and endothelial P-selectin contribute to lesion development; however, endothelial P-selectin plays a crucial role in atherosclerotic lesion growth. 83 Similarly, murine models lacking CD40 in ApoE-deficient mice demonstrate that platelet CD40 promotes atherosclerosis by activation and recruitment of leukocytes to endothelial cells. 84 When platelets lacking CD40 are injected in the circulation of mice, expression of VCAM-1, PECAM (platelet endothelial cell adhesion molecule), VE-cadherin (vascular endothelial cadherin), and P-selectin are decreased on endothelial cells. This decrease in adhesion molecule expression is accompanied by less advanced atherosclerotic plaques with reduced levels of macrophages and neutrophils. 84 Increased expression of platelet CD154, in turn, contributes to accelerated atherosclerosis by increasing platelet platelet interactions and inflammatory response through mediation of platelet endothelium and platelet leukocyte interactions. 85 In addition, platelet CD154 suppresses Treg cell recruitment leading to accelerated atherosclerosis. 85 Platelets and Metastatic Cells in the Circulation Platelets contribute to the increased thrombotic risk of cancer patients. High platelet count is associated with decreased survival in various malignancies, and thrombocytopenia, in turn, is associated with decreased metastatic potential of tumors. Entry of metastatic cancer cells into the blood stream leads to platelet activation. Metastatic cells express PSGL1 and secrete high levels of tissue factor. 33 Platelet cancer cell interactions and cancer-induced thrombi formation in the vasculature may inhibit recognition and elimination by the immune system. In addition, the thrombi formation can lead to tethering of the cancer cell to the endothelium, promoting survival and invasive potential. More recent studies in mice have proposed that, as a consequence of interaction with tumor cells, platelet signals recruit granulocytes to the tumor, thereby, guiding the formation of an early prometastatic microenvironment. Depletion of platelets by cell-specific depleting antibodies inhibits granulocyte recruitment to metastatic cells in lungs, and depletion of granulocytes leads to fewer metastases and reduced metastatic progression. 26 Platelet-derived TGF-β is an important factor contributing to metastasis. In vitro studies have shown that thrombinmediated platelet-derived TGF-β can alter NK-cell antitumor reactivity. 25 In vivo studies eliminating TGF-β specifically from platelets have proposed that this cytokine, when originating from platelets, is solely responsible for inducing an invasive epithelial mesenchymal transition to metastasis. 27 These studies used the PF4Cre murine model which, at the time, was believed to eliminate TGF-β only from the megakaryocyte/ platelet lineage. 27 We have since learned that a subset of epithelial cells can express PF4 86 in addition to activated CD4 + and CD8 + T cells. 17 These studies suggest that other tissue-resident cells or epithelial cells themselves can contribute to the invasive potential of cancer cells mediated by TGF-β. The role of the platelet-mediated increase in metastatic potential through

8 344 Circulation Research January 19, 2018 TGF-β is further complicated because thrombin stimulation also leads to the release of PF4 from platelet α-granules. 17 PF4, in turn, inhibits TGF-β signaling in a SMAD2/3 (Sma- and Mad-related protein)-dependent manner. 17 Regardless of TGFβ signaling, metastatic cancer cells entering the bloodstream hijack the hemostatic function of platelets by releasing tissue factor and, consequently, generate thrombin and initiate thrombus formation and neutrophil recruitment. The formed thrombi can attach to the endothelium and promote viability and metastatic potential of the cancer cell. Platelets in Bacterial Infections During bacterial infections, platelets actively mediate the host response through interactions with circulating leukocytes. In addition to PF4 and RANTES, platelet α-granules contain various antimicrobial compounds such as platelet CTAP-3 (connective tissue activating peptide 3), platelet basic protein, Tβ-4 (thymosin β-4), and fibrinopeptide (A and B). 87,88 These platelet-derived antimicrobial compounds are known to target various bacterial organisms (Figure 3). 4,89,90 Our understanding of how platelets mediate these interactions in the circulation is still developing. Platelets can interact with various strains of bacteria including the Staphylococci family, Neisseria gonorrhoeae, Porphyromonas gingivalis, and Helicobacter pylori. 89,91,92 Platelets adhere and aggregate around bacteria, typically using their GP2b/3a, FcγRIIa (Fc fragment of IgG receptor IIa), and IgG receptors in conjunction with fibrinogen or fibronectin. 14,93 After platelets interact with bacteria, they release antimicrobial compounds contained in their granules. S aureus α-toxin mediates release of β-defensin from platelets, and, consequently, β-defensin induces NET formation. 28 Released PF4 can bind to Gram-negative bacteria, leading to the exposure of PF4/heparin-like epitopes. Exposed PF4 epitopes, in turn, increase antibody binding to the bacterial surface leading to the opsonization and possibly phagocytosis of bacteria by neutrophils. 18,19 This mechanism is particularly important because some Gramnegative bacteria, such as Yersinia pestis, are poorly recognized by the mammalian TLR4. 94,95 In vivo studies using another Gram-negative bacteria, P gingivalis, show that, during infection, platelets interact with neutrophils forming heterotypic aggregates in a TLR2-dependent manner and TLR2 can also promote platelet aggregation. 57 This study provides evidence that, by recognizing bacterial components, platelets can activate platelet thrombotic and/or inflammatory pathways. Studies using Listeria monocytogenes have recently shed light on the role of platelets in inducing adaptive immunity during bacterial infection. Platelet binding to intravascular bacteria leads to slowing of the cytotoxic clearance of the pathogen, keeping bacteria available for induction of an adaptive immune response by splenic CD8 + T cells. 12 This platelet-mediated process is dependent on the opsonization of bacteria by complement C3, subsequent platelet binding by GP1b, and the capture of the bacteria platelet aggregates by cells of the immune system. 12 Platelet bacteria interactions in the presence of C3 lead to different kinetics of clearance between free (fast clearance) and platelet-bound (slow clearance) bacteria. This observation is true for Gram-positive strains, such as L monocytogenes, Bacillus subtilis, Enterococcus faecalis, and Staphylococcus epidermidis, and for Gram-negative strains such as Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae. 12 This study suggests a sophisticated role for platelets in balancing clearance of bacteria and induction of adaptive immunity. Platelets in Viral Infections Platelets interact with various types of viruses. Viral infections are often associated with thrombocytopenia and in some cases thrombosis. Viruses can be simply classified as those with a DNA or RNA genome. RNA viruses are further subdivided into double-stranded and single-stranded viruses. DNA viruses from the herpes viral family, such as herpes simplex virus type 1, cytomegalovirus, and vaccinia, have been found associated with platelets, but it is unclear if they can be internalized by platelets RNA viruses such as HIV, hepatitis C virus, dengue, influenza, coxsackievirus B, and encephalomyocarditis virus are much smaller in size and can be internalized by platelets. 34, Here, we have focused on the interaction of virally activated platelets with circulating cells. In general, platelet function during viral infection can be beneficial or detrimental to the host depending on the length of time post-infection. Human cytomegalovirus binds to platelet TLR2, resulting in release of CD154, IL-1β, and VEGF (vascular endothelial growth factor; Figure 3). Human cytomegalovirus activated platelets do not show increased aggregation or adhesive potential but rapidly form platelet neutrophil heterotypic aggregates. In addition to their interactions with neutrophils, human cytomegalovirus platelets also exhibit increased interaction with monocytes, B cells, T cells, and DCs, suggesting a multifactorial interaction with the innate and adaptive immune systems and induction of proangiogenic responses. 96 In vitro studies using vaccinia virus show that ADP-, collagen-, or thrombin-mediated aggregation in isolated platelets is inhibited by the virus, but there is an increase in serotonin release 98 (Figure 3). In vivo studies, however, show that infection with vaccinia leads to fatal intravascular coagulation at 24 hours post-infection. 104 In vitro studies using herpes simplex virus type 1 infected human umbilical vein endothelial cells show platelet binding because of increased thrombin generation from the damaged cells. 105 These observations suggest that other circulating or vascular cells can contribute to the overall prothrombotic balance. RNA virus infections exhibit more diverse platelet responses. Many RNA viruses cause various degrees of thrombocytopenia at different stages of infection. In the setting of murine encephalomyocarditis virus infection, platelets interact with neutrophils to form heterotypic aggregates as early as 1 hour post infection, and this leads to a sudden drop in platelet count. This drop also occurs at 24 hours post-infection. Elimination of platelets before encephalomyocarditis virus infection leads to reduced survival similar to mice deficient in TLR7. 34 In humans, infection with dengue, an RNA virus, is characterized by profound hemorrhagic fever and thrombocytopenia. In a rhesus macaque model, dengue viral infection leads to platelet monocyte interactions at 24 hours post-infection, whereas platelet neutrophil interactions peak at 3 days post-infection. 106 In this study, blood contained monocytes that had engulfed platelets containing dengue virus. 106 Increased permeability of the endothelium during dengue infection is also facilitated by platelets. Dengue infection mediates assembly of the NLPR3 (NOD-like

9 Koupenova et al Platelets and Vascular Communication 345 Figure 3. Platelet and circulating cell interactions during infection initiate the innate or adaptive immune response. Platelets achieve cell-to-cell communication during bacterial or viral infection either by direct interaction with white blood cells (WBCs) through surface expression of platelet proteins or through indirect protein release from their α- or δ-granules. Encephalomyocarditis virus (EMCV) activated platelets interact with neutrophils in a TLR7 (Toll-like receptor 7)-dependent manner. Coxsackievirus B (CVB) activated platelets bind to neutrophils in a phosphatidylserine (PS)-dependent manner. Dengue and influenza increase microparticle release; denguemediated microparticles contain IL-1β (interleukin 1β). Human cytomegalovirus (HCMV) activated platelets interact with neutrophils, monocytes, B cells, T cells, and dendritic cells (DCs), suggesting activation of innate and adaptive immune responses. Vaccinia-bound platelets have reduced aggregation potential in the presence of ADP, collagen, or thrombin. The specific pathways by which platelets respond to herpes simplex virus (HSV) 1 or HSV2 are currently unknown. During bacterial infection, platelet interactions with complement C3 opsonized bacteria through GP1b (glycoprotein 1b; CD42) lead to slowing of bacterial clearance. DCs recognize the platelet bacterial complexes, thereby inducing adaptive immunity. These platelet bacterial interactions are true for Gram-positive or Gram-negative bacteria. 5HT includes serotonin; and VEGF, vascular endothelial growth factor. receptor family, pyrin domain containing 3) inflammasome and activation of Caspase-1 in platelets. Caspase-1, in turn, alters secretion of IL-1β rich microparticles. 107 Platelet monocyte interactions are also increased in people infected with HIV1 or influenza 108,109 and are influenced by inflammatory stimuli in a COX (cyclooxygenase)-dependent manner. 110 During infection with coxsackievirus B, platelets form aggregates with the neutrophil population and directly improve the outcome of coxsackievirus B induced myocarditis. 103 Coxsackievirus B is internalized by platelets causing phosphatidylserine exposure on the platelet surface without affecting apoptosis. 103 The virus cannot replicate in platelets, and platelet depletion causes an increased viral load in heart tissue and increased myocarditis. 103 Influenza infection illustrates the potential for dysregulation of the hemostatic-inflammatory balance. In patients infected with influenza H1N1 virus, there is an elevation of platelet monocyte aggregates and a 2-fold increase in platelet aggregation potential as measured by PAC1 (pituitary adenylate cyclase-activating polypeptide type I receptor) incorporation 111 (Figure 3). As the infection progresses, coagulation potential increases, as evidenced by the elevation of macrovesicle tissue factor activity. 112 In addition, in nonsurvivor influenza-infected patients, there is a trend toward lowered platelet and increased white blood cell counts. 112 H1N1 activates human platelets independently through both FcγRIIA signaling and through thrombin generation, 15 whereas H1N1 platelet interaction increases the expression of the active form of the fibrinogen receptor GP2b/3a, arachidonic acid metabolism, and microparticle release. 15 Studies using a lethal dose of the H1N1/PR8 strain in C57BL/6J mice suggest that, in critical nonsurvivor settings, platelet degranulation and activation contribute to

10 346 Circulation Research January 19, 2018 lung inflammation and reduced survival. 113 In summary, platelets contribute to the overall procoagulant and inflammatory states during influenza infection, but it is unclear if they are necessary for overall immunity and vascular health. Platelets in Plasmodium Parasite (Malaria) Infection There are 6 plasmodium species that can infect humans and lead to development of malaria. The role of platelets in Plasmodium parasite infection changes as the infection progresses. Studies using noninflammatory malaria with Plasmodium falciparum or Plasmodium chabaudi show that platelets kill the parasite inside of the red blood cells by secreting PF4. 20 PF4, in turn, interacts with the Duffy antigen on red blood cells through an unknown mechanism and mediates survival of infection. 21 Although these studies suggest a protective role during early infection, a model of cerebral malaria using P berghei ANKA (PbA) irbcs clone 2.34 concludes that platelets play multifactorial roles depending on the pathogenic stage of infection. 22 Platelets in this case were activated throughout the first 3 days of infection as measured by an increase in circulating PF4. This early activation of platelets is protective possibly because of a platelet-dependent reduction in parasite burden and leukocyte recruitment through an increase of the IL-1β pool. 22 As infection progresses, however, platelet activation, possibly mediated by the overall immune response and tissue factor release, leads to microvascular thrombosis as found in the postmortem brains of patients with cerebral malaria. 4,114 Platelets and RNA Transfer The ability of platelets to communicate with other vascular cells through receptor signaling, direct protein interactions, or released granule content has long been understood. More recently, other methods of platelet vascular communication have been described including RNA transfer. Bidirectional RNA transmission is illustrated by transfer of platelet-derived transcripts to leukocytes or endothelial cells and by the uptake of leukocyte- or vascular-derived transcripts into platelets 115 (Figure 4). Coculture of platelet-like particles with either a human umbilical vein endothelial cell line or a human monocytic cell line (THP-1) demonstrates functional transfer of plateletderived mrna transcripts. 115 Endogenous analysis of THP-1 cells post platelet-like particle incubation reveals an increased expression of α, γ, and ε globin genes as a consequence of RNA transfer. 115 Confirmation of this phenomenon in vivo using mouse infusion experiments demonstrates that platelets carrying TLR2 mrna can transfer their transcripts to mice with TLR2-deficient blood mononuclear leukocytes. 115 Analysis of human platelets post-coincubation with human umbilical vein endothelial cells and THP-1 cells reveals that platelets can also take up transcripts from surrounding cells. There are distinct changes in expression of platelet transcripts including those associated with platelet vascular adhesion and apoptosis. 116 Platelet RNA uptake has been confirmed by the presence of TLR2 in TLR2-negative platelets post-infusion of TLR2-positive leukocytes (peripheral blood mononuclear cells) into TLR2 knockout mice. 116 Interestingly, Figure 4. Platelet vascular and circulating cellular communication by nonprotein-dependent mechanisms. Platelets and plateletlike particles (PLPs) have been shown to communicate with vascular cells in several protein-independent manners, specifically through mitochondrial and RNA transfer. RNA can be transferred from platelets to endothelial cells, monocytes, macrophages, and cancer cells. Inversely, RNA can be taken up by platelets from endothelial cells, monocytes, and cancer cells. Mitochondrial transfer from platelets to neutrophils can also occur. Microparticles mediate a significant number of these communication pathways.